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1.
Detection by low-temperature magnetic circular-dichroism spectroscopy of optical absorption bands due to molybdenum (V) in the form of xanthine oxidase giving the Desulpho Inhibited e.p.r. signal.
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The magnetic circular-dichroism (m.c.d.) spectra in the temperature range 1.5-100 K and the electronic absorption spectra at 4.2 and 295 K were measured for a number of desulpho xanthine oxidase derivatives. There were no significant differences between the absorption spectra that could be attributed to molybdenum. However, the visible-region m.c.d. spectrum of the ethanediol-treated metalloprotein (which gives rise to the Desulpho Inhibited e.p.r. signal) contained features assignable to Mo(V) absorption bands. This is the first report of the detection of optical bands of Mo(V) in an enzyme in the presence of other chromophoric centres. 相似文献
2.
The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy. 总被引:1,自引:0,他引:1
Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies. 相似文献
3.
The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements. 相似文献
4.
The refolding of rabbit muscle pyruvate kinase after denaturation by guanidine hydrochloride was studied. On dilution of the denaturing agent, enzyme activity is only partially regained. The extent of regain of activity is dependent on protein concentration, showing a marked decrease at higher concentrations. The failure to regain complete activity appears to be related to the formation of inactive aggregates, which can be separated from active enzyme by gel filtration. Insoluble aggregates can be partially re-activated after solubilization in guanidine hydrochloride. Changes in the circular-dichroism and fluorescence spectra during refolding suggest that a partially folded, inactive species is formed rapidly; this differs from native enzyme in being more susceptible to proteolysis by trypsin. 相似文献
5.
Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra deconvolution 总被引:2,自引:0,他引:2
The results are presented for the deconvolution of IR spectra of disaccharides and polysaccharides with alpha and beta configurations of the 1 --> 4 glycosidic linkage (maltose, cellobiose, amylose, and cellulose), as well as of their corresponding monosaccharides (alpha- and beta-D-glucose) in the 1200-920 cm(-1) frequency range. It is established that a characteristic of di- and polysaccharides with 1 --> 4 glycosidic linkage is the appearance of new absorption bands in the 1175-1140 cm(-1) spectral range, as opposed to the IR spectra of monosaccharides. This can be a spectroscopic manifestation of the glycosidic linkage formation. In the 1000-970 cm(-1) frequency range, absorption bands, which are not observed in the monomer spectrum, are separated as a result of the deconvolution of the IR spectra of cellobiose and cellulose. The number of bands in this range remains unchanged for maltose and amylose, as compared to the monomer spectra. It is shown that the application of the method of deconvolution leads to a considerable enhancement in the resolution of the absorption bands in the IR spectra of mono-, di-, and polysaccharides. 相似文献
6.
Iso-cytochrome c species from baker''s yeast. Analysis of their circular-dichroism spectra. 总被引:1,自引:0,他引:1
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The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm. In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c. From the spectra in the u.v. region the latter appears, however, to be more helical. It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70. 相似文献
7.
The circular-dichroism (CD) spectra of beta-lactamases I and II from Bacillus cereus 569/H are reported, along with that of the beta-lactamase II free from carbohydrate. The results show that carbohydrate makes an appreciable contribution to the optical activity of beta-lactamase II in the far-ultraviolet, and that removal of carbohydrate greatly affects the optical activity of several aromatic side chains of the protein moiety. Both tyrosyl and tryptophanyl residues are affected, showing that some of these residues must be near to the surface of the protein moiety, close to the site of attachment of the carbohydrate. Although the far-ultraviolet CD spectrum of beta-lactamase II resembles that of a protein containing some beta-structure, it can be shown that this is a consequence of the optical activity of carbohydrate in this region of the spectrum, and that the protein is likely to contain alpha-helix rather than beta-pleated sheet structure. The overall structures of the protein components of beta-lactamases I and II are similar, but not identical, as shown by the dissimilarity of the CD spectra when calculated on a mean residue basis. 相似文献
8.
The circular dichroism (CD) of synthetic mono- and oligosaccharides that represent the terminal, non-reducing group of O-antigens of Vibrio cholerae O1 from the subtypes Ogawa and Inaba was measured in various solvents. We found differences in the CD of the monosaccharides of these subtypes that decrease with increasing chain lengths of the oligosaccharides. The differences can be explained by different orientations of the N-acyl side chain of the terminal monosaccharides. The linear relationship of ellipticity versus the number of residues in an oligosaccharide chain follows the principle of optical superposition. This, together with a similar contribution by internal units to the overall ellipticity, suggests an identical, regular conformation of oligosaccharide fragments of both Ogawa and Inaba series. 相似文献
9.
The allosteric mechanism of bovine liver glutamate dehydrogenase. Evidence from circular-dichroism studies for a conformational change in the ternary complex enzyme-(oxidized nicotinamide-adenine dinucleotide)-glutarate.
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1. Computer averaging of multiple scans was used to refine the circular dichroism spectrum of bovine liver glutamate dehydrogenase, revealing well-defined structure in the aromatic region. 2. The circular dichroism of NAD+ bound to glutamate dehydrogenase is strongly negative at 260nm, probably owing to immobilization of the adenosine moiety. Loss of the characteristic adenine-nicotinamide interaction suggests that the coenzyme is bound in an unstacked conformation. 3. Glutarate and succinate, substrate analogues that are both inhibitors competitive with glutamate, do not significantly perturb the circular-dichroism spectrum of the enzyme in the absence of NAD+. 4. In the presence of NAD+, 150nM-succinate decreases the negative circular dichroism corresponding to bound coenzyme, but does not affect the protein circular dichroism. However, ISOmM-glutarate causes profound alternations of the circular-dichroism spectra of the bound NAD+ and of the enzyme, indicative of a protein conformational change. This direct evidence of conformational change specifically promoted by C5 dicarboxylates confirms the previous inference from protection studies. 5. The conformational change is discussed in relation to the allosteric mechanism of glutamate dehydrogenase. 相似文献
10.
Properties of protein-chlorophyll complexes from pea (Pisum sativum L.) leaves. The organization of chlorophyll.
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Chlorophyll-protein-detergent complexes were prepared from pea chloroplasts by using sodium dodecylbenzenesulphonate and polyacrylamide-gel electrophoresis. Circular-dichroism spectra showed that complex CPI has a dimeric arrangement of chlorophyll a, with additional weaker interactions. Ellipticities were determined for both complexes and for purified chlorophylls in solution, and it is argued that the circular dichroism of complex CPII is derived from chlorophyll-protein interaction rather than from interaction between chlorophylls a and b. The detergent could be removed from the complexes by using urea and gel filtration, leaving the chlorophyll-protein in solution, although in each case a diminished ellipticity indicated some loss of organization. Three-peaked circular-dichroism spectra of chloroplast fragments before and after addition of detergent were compared with a curve obtained by summing graphically the spectra of complexes CPI, CPII and the free-pigment fraction. There was good correspondence at 650 nm, and the longer-wavelength peaks agreed in form and magnitude, but with discrepancies in position. It was concluded that complexes CPI and CPII pre-exist in the original material, but that there is an environmental effect which is destroyed when the complexes are extracted. 相似文献
11.
The 2-aminothiophenol-based fluorometric assay of Nakano et al. (1973, J. Pharm. Soc. Jpn. 93, 350-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 50 ng of carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates. 相似文献
12.
Furuki T 《Carbohydrate research》2000,323(1-4):185-191
Thermal behavior at subzero temperatures has been investigated for aqueous solutions of various monosaccharides. The heat of fusion of ice measured with differential scanning calorimetry has given linear plots against sugar concentration (wt.%), from which the amount of unfrozen water, Uw, has been determined for each monosaccharide. The results for Uw are analyzed by employing, as a measure of hydration characteristics, known physico-chemical properties of aqueous monosaccharides, such as partial molar compressibilities, etc. It was revealed that the anti-freeze characteristics of carbohydrates depend on their stereochemistry. More water remains unfrozen in the aqueous solutions of carbohydrates having poorer compatibility with the three-dimensional hydrogen-bond network of water. Monosaccharides studied can be subdivided into three groups according to the extent of the anti-freeze effect. These results are rationalized in terms of a modified stereospecific hydration model. 相似文献
13.
Evidence for similar conformational changes in α2-macroglobulin on reaction with primary amines or proteolytic enzymes
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Reactions of alpha(2)-macroglobulin (alpha(2)M) with primary amines (ammonium chloride, methylammonium chloride and ethylammonium chloride) or proteolytic enzymes (trypsin, chymotrypsin and thrombin) resulted in changes of the absorption, fluorescence and circular-dichroism spectra and of the sedimentation coefficient of the inhibitor. All physico-chemical changes caused by the inactivation of alpha(2)M by the amines were identical with, or highly similar to, those induced by the formation of the enzyme-inhibitor complexes. This suggests that similar conformational changes of the inhibitor occur in the two types of reactions. The frictional ratio, calculated from the increase in sedimentation coefficient, decreased from 1.67 for untreated alpha(2)M to 1.57 for the amine- or proteinase-treated inhibitor. This change is due to a decrease in either asymmetry or hydration of the protein, resulting in a slightly smaller hydrodynamic volume. The circular-dichroism analyses indicated that the reaction of alpha(2)M with either amines or proteinases is accompanied by a loss of the small amount (about 5%) of alpha-helix of the untreated protein. The changes of u.v. absorption and fluorescence suggested that about one out of the eight to ten tryptophan residues of each alpha(2)M subunit is buried as a result of the conformational change. All spectroscopic and hydrodynamic changes that were observed are compatible with a spatial rearrangement of the subunits of alpha(2)M, as implicated by the ;trap' hypothesis for the mechanism of inhibition of proteinases. However, a conformational change involving a decrease in the hydrodynamic volume of each subunit cannot be excluded. 相似文献
14.
Three isotope-edited n.m.r. methods have been applied to selectively 13C-substituted monosaccharides and nucleosides to simplify their spectra and/or measure 1H-1H, 13C-1H, or 13H-13C spin-couplings detected via the labeled site. 1D INADEQUATE spectra allowed the selective detection of the natural-abundance carbons that are spin-coupled to the labeled carbon, and adjustment of the mixing time permitted further discrimination between one-bond and longer-range 13C-13C coupling pathways. Geminal and vicinal 13C-1H coupling constants were determined from the analysis of 1H-1H COSY cross-peaks for those protons coupled to the labeled carbon. Long-range 13C-(HETCOR) and 1H-detected (HMBC) 13C-1H chemical-shift correlation spectra permitted the selective observation of those protons coupled to the labeled site, and JH,H values were measured from data projections. The implications of these methods for structural studies of more complex systems is briefly discussed. 相似文献
15.
The nature of the circular-dichoric spectra of complexes between ribonuclease A and nucleotides.
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S M Dudkin L V Karabashyan M Y Karpeisky S N Mikhailov N S Padyukova V G Sakharovsky 《The Biochemical journal》1977,167(3):749-757
The circular-dichroism and proton-magnetic-resonance spectra of complexes of ribonuclease A with dihydrouridine 3'-phosphate, 2'- and 3'-CMP, arabinosyl-3'-CMP, 1-(2-hydroxyethyl)cytosine 2'-phosphate and 1-(3-hydroxypropyl)cytosine 3'-phosphate were studied. Comparison of the results shows that non-additivity of the circular-dichroic spectrum of an enzyme-nucleotide complex may be due to: (a), alteration of the circular dichroic spectrum of the nucleotide under the influence of the asymmetric protein matrix (induced dichroism), and (b) a change in the nucleotide conformation. The contribution of each of the two factors was estimated to calculate the circular-dichoroic spectra of 2'-CMP and 3'-CMP in complex with ribonuclease A. 3'-CMP in this complex was characterized by negative circular dichroism in the long-wavelength absorption band of the nucleotide, whereas 2'-CMP was characterized by positive circular dichroism. Since both nucleotides in the complex are known to be in an anti conformation, it follows that even small changes in the conformation considerably modify the circular-dichroic spectrum of the nucleotide in complex with the enzyme. 相似文献
16.
Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of =0.08% and =0.02%, respectively, which allowed determination of the constituent parts of the detected ions in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids. Molecular species were found based on ceramide with mainly sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), and with a total number of monosaccharides ranging from 11 (neutral, m/z = 2605 for [M+Na](+)) to 41 (one sialic acid, m/z = 8057 for [M-H](-)). The saccharide composition obtained supported a successively extended and branched N -acetyllactosamine core with substitutions of fucoses (0 up to 8) and sialic acid (0 to 1). The reliable molecular analysis of large oligosaccharides linked to ceramide using this approach will be of great help for the further structure analysis in order to define the epitope for the sialic acid-dependent binding by the bacterium. 相似文献
17.
Two procedures for analyzing overlapping optical spectra of mixtures of pyridine hemochromes are described, and extinction coefficients of pyridine hemochromes are provided for use with these methods. In the first procedure, absorbance is measured at a number of wavelengths equal to the number of components to be analyzed. This is the minimum amount of spectral data from which the concentration of each species can be calculated. In the second procedure, absorbance is measured at a number of wavelengths greater than the number of components to be analyzed. This redundancy of information makes it impossible to fit spectra which contain contributions from additional components, unless the spectra of the additional components are equal to linear combinations of the spectra of the species being analyzed. These two procedures are generally applicable to analyses of absolute or difference spectra of mixtures of components obeying Beer's law. The sensitivity to error in the absorbance measurements is only slightly greater than that for measuring a pure component at a single wavelength. 相似文献
18.
The term 'glycomics' describes the scientific attempt to identify and study all the glycan molecules - the glycome - synthesised by an organism. The aim is to create a cell-by-cell catalogue of glycosyltransferase expression and detected glycan structures. The current status of databases and bioinformatics tools, which are still in their infancy, is reviewed. The structures of glycans as secondary gene products cannot be easily predicted from the DNA sequence. Glycan sequences cannot be described by a simple linear one-letter code as each pair of monosaccharides can be linked in several ways and branched structures can be formed. Few of the bioinformatics algorithms developed for genomics/proteomics can be directly adapted for glycomics. The development of algorithms, which allow a rapid, automatic interpretation of mass spectra to identify glycan structures is currently the most active field of research. The lack of generally accepted ways to normalise glycan structures and exchange glycan formats hampers an efficient cross-linking and the automatic exchange of distributed data. The upcoming glycomics should accept that unrestricted dissemination of scientific data accelerates scientific findings and initiates a number of new initiatives to explore the data. 相似文献
19.
Positive ion fast atom bombardment mass spectrometry appears to be a very useful method for the determination of molecular weight and composition of underivatized di- and trisaccharides. Information on the identity of the monosaccharide units can be obtained from the metastable ion and collisional activation spectra of selected ions. The type of linkage between the monosaccharides is reflected in some spectral characteristics, but the differences are relatively small and do not always allow an unambiguous identification. The position of a fructose unit in a trisaccharide molecule is shown by the collisional activation spectra of the [M + H]+ ion, as an anhydrofructose molecule is easily eliminated from the ions in which fructose is in a terminal position. 相似文献
20.
Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases. 总被引:5,自引:0,他引:5
Florence Goubet Peter Jackson Michael J Deery Paul Dupree 《Analytical biochemistry》2002,300(1):53-68
A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative. 相似文献